This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-EY-101: Protein misfolding in degenerative diseases of the eye. Identifying therapeutic pharmacological agents/drugs that prevent the misfolding/aggregation of proteins could provide new tools for treating these diseases. Although degeneration of the cells in the eye underlies various pathologies including cataracts, age related macular degeneration and glaucoma, currently there are no approved therapeutic agents for treating cellular degeneration associated with these disorders. One difficulty underlying the development of such therapeutic agents is the complex etiology of these degenerative disorders, wherein multiple cellular pathways are altered. Thus, a therapeutic candidate would ideally target multiple stress pathways underlying cellular degeneration. Our project addresses a critical dilemma for protein-based therapy in the eye: how to provide therapeutic proteins for long-term therapy while at the same time minimizing risks and costs associated with repeated intraocular injections. It is likely that drugs intended for treating degenerative disorders of the eye should be administered from the time of diagnosis onwards. Currently approved protein therapeutics administered in the eye have a relatively short half-life (<10 days), which necessitates repeated injections to the eye approximately every 6 weeks. Thus, there is a need for slow release systems for prolonged delivery of macromolecules to the eye. Technology for targeted delivery of macromolecules to degenerating cells of the eye is also an important goal. Protein drugs, besides being susceptible to proteolytic enzymes that are ubiquitous, do not enter target cells efficiently due to their large molecular size. In order to overcome this limitation, functionalized nanoparticles encapsulating the protein drugs need to be developed. The small heat shock proteins (sHSP) proteins aA-crystallin and aB-crystallin have excellent potential as therapeutic proteins against some of the most common eye diseases seen in the clinic today. Like other members of the sHSP family, [unreadable]-crystallin oligomers display chaperone-like activity defined by their ability to prevent aggregation of other proteins. Outside the eye, [unreadable]-crystallin has been demonstrated to co-localize with protein aggregates implicated in the pathogenesis of a variety of diseases, including fibrillary bundles associated with neurodegenerative and autoimmune diseases. Introduction of sHSPs via plasmid or viral expression vectors has been demonstrated to rescue phenotypic markers in a variety of disease models, including retinitis pigmentosa (RP) and aggregation-prone cataract mutants. Consistent with the emphasis on translational science in this challenge announcement, we propose to develop sHSP-based therapeutics to prevent and/or reverse the formation of aggregates implicated in the pathogenesis of cataract and RP. Our overarching hypothesis is that developing an efficient method for delivery of sHSPs to the eye will provide a novel new strategy for treatment of a broad range of eye conditions caused by genetic disorders and metabolic stress. Our proposal is organized around two major goals. In the first aim, we will develop and optimize various technologies for preparing nanoparticles loaded with human sHSP. In the second aim, we will test the effectiveness of sHSP delivered via nanoparticles at suppressing and/or reversing high molecular weight aggregates formed from mutant proteins associated with RP and cataracts. Specific Aim 1: Develop a series of sHSP nanomedicine formulations and optimize for sustained slow release into the eye. We will pursue four different technologies for generating slow release of sHSP complexes into the eye. Combinations of wild type and "activated" sHSP subunits derived from human sHSP genes will be incorporated into nanoparticles. Studies will be carried out to optimize protein release and tissue penetration from different nanoparticle formulations. Specific Aim 2: Test the hypothesis that sHSP can suppress formation of insoluble high molecular weight oligomeric complexes initiated by aggregation-prone rhodopsin and lens crystallin mutants. Nanoparticle formulations that demonstrate efficacy in tissue culture models of RP and cataract will be tested in animal models of protein aggregation disease, including the P23H transgenic rat model of RP and the aA-crystallin knock out mouse model of cataract. PUBLIC HEALTH RELEVANCE: This project seeks to develop new drugs to prevent or reverse blinding diseases such as cataract and retinitis pigmentosa, which are associated with aggregation of proteins. Protein aggregation diseases account for some of the most common degenerative diseases of the eye. Our proposed studies could substantially improve the quality of life of affected patients by preventing vision loss and associated financial burdens due to lost productivity and increased medical expenses.